The Problem: Air pollution enhances susceptibility to influenza infection, but mechanisms are poorly defined. The potential public health impact is high, since future influenza epidemics and pandemics will involve urban areas with high levels of pollution exacerbating the spread and severity of infection. The Opportunity: Genome-wide RNAi screens allow discovery of host genes and cellular functions essential for infection by influenza viruses. We reason that the RNAi functional genetic screening approach could also provide new insights into how air pollution exposures modify susceptibility to influenza. In pilot studies, we established that the soluble form of residual oil fly ash (ROFA) a surrogate for urban air pollution, enhances influenza virus-induced cell death of lung epithelium. Initial use of this cell-based assay for functional genetic screening reveals involvement of novel genes and supports feasibility. The goal of this project is to perform RNAi screens to identify critical cellular genes which mediate pollution-enhanced susceptibility to influenza infection. The Solution: Aim 1: Using a novel lentiviral RNAi library, we will perform genome-wide screening to identify genes whose 'knockdown' inhibition results in greater survival after influenza infection alone, diesel exhaust exposure alone and pre-infection exposure to pollutant. Aim 2: Using the same genome-wide screen strategy as in aim 1, we will identify gene sets that mediate enhanced cell death upon exposure to pollutant after influenza infection. Aim 3 will validate a panel of 10 top candidates (selected for magnitude of functional effect, and bioinformatics analysis to prioritize biologic relevance and novelty) by testing for: 1) expression of the target gene by lung epithelial cells, 2) effective knockdown by individual shRNAs, and 3) replication of the phenotype (increased resistance to influenza despite pollutant exposure) by individual gene knockdown; 4) similar assays of expression and function in airway epithelium (including primary human lung epithelial cells). Significance: This exploratory project will build on promising pilot data to identify functionally defined genes that mediate air pollutant enhancement of influenza. The results will open new lines of specific mechanistic research in how environmental exposures impact this important infection.